Adenosine is a nucleoside that occurs naturally in mammals, which acts as a ubiquitous biochemical messenger. The heart, for instance, produces and releases adenosine in order to modulate heart rate and coronary vasodilation. Likewise, adenosine is produced in the kidney to modulate essential physiological responses, including glomerular filtration rate (GFR), electrolyte reabsorption, and renin secretion.
Adenosine is known to bind to and activate seven-transmembrane spanning G-protein coupled receptors, thereby eliciting a variety of physiological responses. There are 4 known subtypes of adenosine receptors (i.e., A1, A2A, A2B, and A3), which mediate different, and sometimes opposing, effects. For example, activation of the adenosine A1 receptor, elicits an increase in renal vascular resistance, which leads to a decrease in glomerular filtration rate (GFR), while activation of the adenosine A2A receptor elicits a decrease in renal vascular resistance. Conversely, blockade of the A1 adenosine receptor decreases afferent arteriole pressure, leading to an increase in GFR and urine flow, and sodium excretion. Furthermore, A2A adenosine receptors modulate coronary vasodilation, whereas A2B receptors have been implicated in mast cell activation, asthma, vasodilation, regulation of cell growth, intestinal function, and modulation of neurosecretion (See, Adenosine A2B Receptors as Therapeutic Targets, Drug Dev Res 45:198; Feoktistov et al., Trends Pharmacol Sci 19:148-153 and Ralevic, V and Burnstock, G. (1998), Pharmacological Reviews, Vol. 50: 413-492), and A3 adenosine receptors modulate cell proliferation processes. Two receptor subtypes (A1 and A2A) exhibit affinity for adenosine in the nanomolar range while two other known subtypes A2B and A3 are low-affinity receptors, with affinity for adenosine in the low-micromolar range. A1 and A3 adenosine receptor activation can lead to an inhibition of adenylate cyclase activity, while A2A and A2B activation causes a stimulation of adenylate cyclase.
It has been shown that adenosine, acting at specific cell surface receptors, has the potential to suppress inflammation and that inflammation itself may increase extracellular adenosine levels (Cronstein, et al., 1986, Journal of Clinical Investigation 78:760-770; Cronstein, et al., 1983, Journal of Experimental Medicine 158:1160-1177). Further, it has been demonstrated that adenosine mediates the anti-inflammatory effects of low-dose methotrexate therapy for Rheumatoid Arthritis (Reviewed in Cronstein, 2005, Pharmacol Rev 57:163-172). Exploration of the therapeutic and toxic properties of methotrexate in the treatment of RA has led to a number of other potentially important pre-clinical therapeutic developments. Methotrexate increases giant cell formation from peripheral blood monocytes and that this effect is mediated by adenosine acting at A1 receptors (Merrill, et al., Arth. Rheum. 40:1308-1315). In addition, A2A receptor antagonists promote giant cell formation by diminishing the effect of endogenous adenosine although the A1 receptor-mediated promotion of giant cell formation appears to dominate.
Cronstein, U.S. Pat. No. 7,795,427 describes the use of agents that block adenosine A1 receptor antagonists to diminish osteoclast function and thereby prevent the development of osteoporosis. U.S. Pat. No. 8,183,225 describes the activation of adenosine A2A receptors as inhibiting osteoclast formation and function, and use of adenosine A2A receptor agonists to prevent wear particle-induced bone resorption. In all of these actions adenosine receptor blockade or activation was directed solely at preventing bone resorption. Interestingly, these studies do not demonstrate that either adenosine A1 or A2A receptors affect the formation or function of osteoblasts. U.S. Ser. No. 14/380,238 describes the use of modulators of an adenosine receptor, including agonists of an adenosine A2A receptor and antagonists of an A1 receptor, to stimulate bone regeneration and stimulate and promote differentiation and activation of osteoblasts as well as potentially inhibit bone resorption and inhibit differentiation and stimulation of osteoclasts.
The prior art also teaches use of adenosine receptor agonists and antagonists or dipyridamole in the regulation of osteoblast differentiation, proliferation and function. Moreover, any proposed use of dipyridamole described in the prior art is to increase adenosine to stimulate adenosine A2B receptors to stimulate osteoblast production of bone matrix and inhibit IL-6 production or increase production of osteoprotegerin. (See, e.g., Kara et al., The FASEB Journal 2010; 24:2325-2333; Kara et al., Arthritis and Rheumatism 2010; 62:534-541; Russell et al., Calcif Tissue Int 2007; 81:316-326; Evans et al., J Bone Miner Res 2006; 21:228-236; Costa et al., Journal of Cellular Physiology 2011; 226:1353-1366).
Adenosine is an endogenously produced physiologic regulator whose intracellular and extracellular concentration is tightly regulated by oxygen consumption, cellular stress and mitochondrial functionality. Extracellular adenosine derives mainly from hydrolysis of ATP (primarily, but not exclusively, by the ectoenzymes CD39 and CD73) and mediates its effects via activation of G protein coupled receptors (A1R, A2AR, A2BR, A3R). Adenosine has long been known to regulate inflammation and immune responses (Hasko et al., Nature Reviews. Drug Discovery 2008; 7:759-770; Hasko et al., Frontiers in Immunology 2013; 4:85), and recent work has demonstrated the importance of adenosine and its receptors in osteoblast, osteoclast, and bone marrow homeostasis (Kara et al., FASEB Journal: official publication of the Federation of American Societies for Experimental Biology 2010; 24:2325-2333; Kara et al., Arthritis and Rheumatism 2010; 62:31534-541; He et al., Front Biosci (Elite Ed) 2011; 3:888-895; He et al., Purinergic Signalling 2012; 8:327-337; Mediero et al., Am J Pathol 2012; 180:775-786; He et al., FASEB Journal: official publication of the Federation of American Societies for Experimental Biology 2013; 27:3446-3454; He et al., British Journal of Pharmacology 2013; 170:1167-1176; Mediero et al., Trends in endocrinology and metabolism: TEM 2013; 24:290-300; Mediero et al., British Journal of Pharmacology 2013; 169:1372-1388; Lerner et al., Acta Physiol Scand 1987; 131:287-296; Shimegi et al., Calcified Tissue International 1996; 58:109-113; Jones et al., Bone 1997; 21:393-399; Shimegi et al., Calcified Tissue International 1998; 62:418-425; Evans et al., Journal of Bone and Mineral Research: the official journal of the American Society for Bone and Mineral Research 2006; 21: 228-236; Russell et al., Calcified tissue international 2007; 81:316-326; Orriss et al., Current opinion in pharmacology 2010; 10:322-330; Costa et al., Journal of cellular physiology 2011; 226:1353-1366; Gartland et al. Front Biosci (Landmark Ed) 2012; 17:16-29). Prior studies have suggested that adenosine receptors also regulate chondrocyte physiology and pathology in response to inflammatory stimuli although the specific receptor(s) involved have not been fully clarified. Removal of endogenous adenosine (by addition of adenosine deaminase) or blockade of A2AR leads to cartilage degradation in equine cartilage explants although equine purine metabolism differs from other species as adenosine deaminase, present in lymphocytes, plasma and extracellular fluid of most species, is not present in horse lymphocytes or serum (Koolpe et al., Arthritis and rheumatism 1999; 42:258-267; Benton et al., Am J Vet Res 2002; 63:204-210; Tesch et al., Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society 2002; 10:34-43; Kono et al., Cell Biochem Funct 2006; 24:103-111; Varani et al., Osteoarthritis and Cartilage/OARS, Osteoarthritis Research Society 2008; 16:292-304; Tesch et al., Osteoarthritis and Cartilage/OARS, Osteoarthritis Research Society 2004; 12:349-359; Hovi et al., Clinical and Experimental Immunology 1976; 23:395-403; Tax et al., Comp Biochem Physiol B 1978; 61:439-441). More recently A3R stimulation was reported to diminish OA development in a chemically induced model of OA35, principally due to the anti-inflammatory effects of A3R agonists.
Mice lacking A2AR were first developed by Chen and colleagues in 1999 and, in general, these mice have few obvious defects (Chen et al., J Neurosci 1999; 19:9192-9200). Interestingly, they do suffer osteopenia as a result of an increase in osteoclast number and function and they respond abnormally to a variety of stressors reflecting the loss of the A2AR. However, as these mice age they have increasing difficulty in grabbing food, walking, and mating, and the diminished mobility of A2AR-deficient mice might be due to intrinsic joint disease.
Osteoarthritis (OA) is the most common type of arthritis, affecting up to 25% of the 3 population over 65, and 12% of all cases may be due to prior joint trauma (Lieberthal et al., Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society 2015; 23:1825-1834; Brown et al., Journal of Orthopaedic Trauma 2006; 20:739-744). Worldwide in its distribution, the incidence of OA increases with age and the resulting pain, loss of joint function and mobility, social isolation, and a broadly reduced quality of life make OA a condition with a high medical and social impact. Current treatment options are less than optimal and do not correct the underlying problem with the result that joint replacement surgery is often the eventual outcome (Mobasheri et al., Current Rheumatology Reports 2013; 15:364).
OA is characterized by changes in every structure in the joint, including cartilage destruction, synovial inflammation, osteophyte formation, enthesophytes, and significant bony changes (Wieland et al., Nature Reviews. Drug Discovery 2005; 4:331-344). The central player in OA is the chondrocyte, which responds to excess mechanical loading by releasing inflammatory mediators and proteolytic enzymes causing further cartilage damages. In addition, age-related inflammation contributes to the pathogenesis of OA (Loeser et al., Nature Reviews. Rheumatology 2016; 12:412-420).
Liposomes are spherical vesicles having at least one lipid bilayer. The liposome can be used as a vehicle for administration of nutrients and pharmaceutical drugs. Liposomes can be prepared by disrupting biological membranes (such as by sonication). Liposomes are most often composed of phospholipids, especially phosphatidylcholine, but may also include other lipids, such as egg phosphatidylethanolamine, so long as they are compatible with lipid bilayer structure. Cevc, Journal of Controlled Release, 1993; 160 (2): 135-146 A liposome design may employ surface ligands for attaching to unhealthy tissue. Torchilin, Advanced Drug Delivery Reviews 2006; 58 (14): 1532-55.
The major types of liposomes are the multilamellar vesicle (MLV, with several lamellar phase lipid bilayers), the small unilamellar liposome vesicle (SUV, with one lipid bilayer), the large unilamellar vesicle (LUV), and the cochleate vesicle. A less desirable form are multivesicular liposomes in which one vesicle contains one or more smaller vesicles.
A liposome has an aqueous solution core surrounded by a hydrophobic membrane, in the form of a lipid bilayer; hydrophilic solutes dissolved in the core cannot readily pass through the bilayer. Hydrophobic chemicals associate with the bilayer. A liposome can be hence loaded with hydrophobic and/or and hydrophilic molecules. To deliver the molecules to a site of action, the lipid bilayer can fuse with other bilayers such as the cell membrane, thus delivering the liposome contents; this is a complex and non-spontaneous event, however. Cevc, Advanced Drug Delivery Reviews, 1993; 38 (3): 207-232 By preparing liposomes in a solution of DNA or drugs (which would normally be unable to diffuse through the membrane) they can be (indiscriminately) delivered past the lipid bilayer, but are then typically distributed non-homogeneously. Barenholz, et al., (2000). Physical chemistry of biological surfaces, Chapter 7: Structure and properties of membranes. New York: Marcel Dekker. pp. 171-241.
Liposomes are used as models for artificial cells. Liposomes can also be designed to deliver drugs in other ways. Liposomes that contain low (or high) pH can be constructed such that dissolved aqueous drugs will be charged in solution (i.e., the pH is outside the drug's pI range). As the pH naturally neutralizes within the liposome (protons can pass through some membranes), the drug will also be neutralized, allowing it to freely pass through a membrane. These liposomes work to deliver drug by diffusion rather than by direct cell fusion.
A similar approach can be exploited in the biodetoxification of drugs by injecting empty liposomes with a transmembrane pH gradient. In this case the vesicles act as sinks to scavenge the drug in the blood circulation and prevent its toxic effect. Bertrand, et al., ACS Nano 4 2000; (12): 7552-8 Another strategy for liposome drug delivery is to target endocytosis events. Liposomes can be made in a particular size range that makes them viable targets for natural macrophage phagocytosis. These liposomes may be digested while in the macrophage's phagosome, thus releasing its drug. Liposomes can also be decorated with opsonins and ligands to activate endocytosis in other cell types. The use of liposomes for transformation or transfection of DNA into a host cell is known as lipofection.
As of 2012, some 13 drugs with liposomal delivery systems have been approved and five additional liposomal drugs were in clinical trials. The clinically approved liposomal drugs include amphotericin B, ctyarabine, daunorubicin, doxorubicin, IRIV vaccine, morphine, verteporfin, proteins SP-B and SP-C, estradiol, vincristine, and PEG.
Liposomes rarely form spontaneously. They typically form after supplying enough energy to a dispersion of (phospho)lipids in a polar solvent, such as water, to break down multilamellar aggregates into oligo- or unilamellar bilayer vesicles. Cevc, Journal of Controlled Release, 1993; 160 (2): 135-146; Barenholz, et al., (2000). Physical chemistry of biological surfaces, Chapter 7: Structure and properties of membranes. New York: Marcel Dekker. pp. 171-241.
Liposomes can hence be created by sonicating a dispersion of amphipatic lipids, such as phospholipids, in water. Low shear rates create multilamellar liposomes. The original aggregates, which have many layers like an onion, thereby form progressively smaller and finally unilamellar liposomes (which are often unstable, owing to their small size and the sonication-created defects). Sonication is generally considered a “gross” method of preparation as it can damage the structure of the drug to be encapsulated. Newer methods such as extrusion and Mozafari method are employed to produce materials for human use. Colas, et al., “Microscopical investigations of nisin-loaded nanoliposomes prepared by Mozafari method and their bacterial targeting”. Micron (Oxford, England: 1993) 2007; 38 (8): 841-7 Using lipids other than phosphatidylcholine can greatly facilitate liposome preparation. Cevc, Journal of Controlled Release, 1993; 160 (2): 135-146.
All publications, patent applications, patents and other reference material mentioned are incorporated by reference in their entirety. In addition, the materials, methods and examples are only illustrative and are not intended to be limiting. The citation of references herein is not to be construed as an admission that the references are prior art to the present invention.